and connected via an additional set of microgrooves. The long length of this set of microgrooves ensures that only axons from this compartment (we refer to this compartment as the “presynaptic” compartment) are able to extend into the perfusion channel. The perfusion channel is 50 μm in width and 100 μm in height (i.e., the same height as the compartments) (Figure 3A). A reservoir (6 mm in diameter) at one end of the channel contains the perfusate and the other end has a smaller opening which is used to connect to efflux tubing and a syringe pump. Withdrawal of the perfusate using the syringe pump results in a negative pressure in the perfusion channel, drawing the perfusate through the channel and preventing diffusion of the perfusate into microgrooves (Figure 3A). To visualize the perfusion, we filled the perfusion reservoir in a neuron-free chamber with Alexa Fluor 488 and withdrew it from the other end using the syringe pump (rate = 100 μl/h) (Figure 3B,C). A uniform fluorescence was rapidly established within the channel; notably, there was no fluorescent signal apparent within the microgrooves. To determine the stability of the perfusion stream and to determine, conclusively, if there is any diffusion
